The Principle of the Redox Activity Expression for FMN-binding Protein Based on its Higher Structure
| Project/Area Number |
14560075
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Osaka City University |
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Principal Investigator |
KITAMURA Masaya Osaka City University, Graduate School of Engineering, Lecturer, 大学院・工学研究科, 講師 (20244634)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2003: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2002: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | FMN / Redox Potential / Protein Engineering / Mutant / X-ray Crystallography / sulfate-reducing Bacteria / Dissociation Constant / Flavodoxin |
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| Research Abstract |
I studied FMN-binding protein (FMN-bp) from the sulfate-reducing bacteria by use of protein-engineering methods. Comparing the amino acid sequence of FMN-bp with that of flavodoxin, which has almost same molecular weight and binds FMN as a prosthetic group, the only Thir-Trp-Asn sequence is the same each other. Using site-directed mutagenesis, 9 mutant FMN-bps were produced by genetic engineering, and were disucussed about the influences of this sequence on the binding of FMN and the redox potential which was measured using safranine T as a mediator. The redox potential of every mutant that changed Trp residue to Ala, Tyr, Phe or His residue shifted rather positive (+8 to +18 mV). This result suggested that Trp residue might not be responsible on redox potential. However, this residue was indicated to be important on binding FMN. On the other hand, the mutant that changed Asn residue to Gin or Asp residue showed little difference on both binding FMN and redox potential. The Asn residue is least conserved in Thr-Trp-Asn and my result indicated that Asn residue was least important in these residues. The redox potential of the mutant that changed Thr residue to Ser or Val residue shifted rather (5 mV) or much (about 60 mV) negative, respectively. This result indicated that it is important on redox potential that this position keeps hydrophilic. Then the mutant that changed Thr residue to Val residue was crystallized and determined the higher structure using x-ray. We obtained the data whose resolution was over 1.5A and now we are proposing its model.
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Report
(3 results)
Research Products
(15 results)
